Microalgal biomass protein enrichment method

ABSTRACT

The invention relates to a method for protein enrichment of a microalga grown under heterotrophic conditions, said microalga being of the  Chlorella  genus, characterized in that the heterotrophic culture comprises a step aimed at limiting the growth of said microalga by means of a deficiency of the fermentation medium in terms of a non-nitrogenous nutritional source.

The present invention relates to a method of protein enrichment ofmicroalgal biomass, said microalga being more particularly of the genusChlorella, even more particularly of the species Chlorella sorokinianaor Chlorella protothecoides.

Algae—macroalgae and microalgae—have a specific but largely unexploredrichness. Their use in the production of food, chemicals and bioenergyremains very rare. They conceal, however, components of great value,whose richness and abundance can be truly appreciated only by the marineanimals that feed on them.

Microalgae are in fact sources of vitamins, lipids, proteins, sugars,pigments and antioxidants.

Algae and microalgae are thus of interest to the industries that usethem to produce dietary supplements, functional foods, cosmetics ormedicines, or for aquaculture.

Microalgae are above all photosynthetic microorganisms which colonizeall biotopes exposed to light.

On an industrial scale, their monoclonal culturing is carried out inphotobioreactors (autotrophic conditions: with light and CO₂) or, forsome, also in fermentors (heterotrophic conditions: in darkness in thepresence of a carbon source).

A few microalgae species are in fact able to grow in the absence oflight: Chlorella, Nitzschia, Cyclotella, Tetraselmis, Crypthecodinium,Schizochytrium.

Furthermore, it is estimated that culturing under heterotrophicconditions costs 1/10 that of culturing under phototrophic conditionsbecause, for the skilled person, heterotrophic conditions make itpossible to:

-   -   use fermentors that are identical to those used for bacteria and        yeasts and that allow all of the culture parameters to be        controlled.    -   produce biomasses in much greater amounts than those obtained by        light-based culturing.

The profitable use of microalgae generally requires control of thefermentation conditions so as to accumulate the components of interest,such as:

-   -   pigments (chlorophyll a, b and c, β-carotene, astaxanthin,        lutein, phycocyanin, xanthophylls, phycoerythrin, etc.)        increasingly in demand for their remarkable antioxidant        properties and for providing natural colors in foods,    -   proteins, in order to optimize their nutritive qualities; or    -   lipids, in order to optimize their fatty acid content (up to        60%, even 80% by weight of their dry matter), in particular for:        -   biofuel applications, but also        -   human or animal food applications, when the selected            microalgae produce so-called “essential” (i.e., provided in            the diet because they are not naturally produced by humans            or animals) polyunsaturated fatty acids, or PUFAs.

To arrive at this result, first high cell density (HCD) fermentationmethods were thus thoroughly investigated, so as to obtain maximumyields and productions of proteins or lipids.

The objective of these HCD cultures was to obtain the desired product inthe highest possible concentration in the shortest amount of time.

This precept is verified, for example, for the biosynthesis ofastaxanthin by Chlorella zofingiensis, where the growth of the microalgaproved to be directly correlated with the production of this compound(Wang and Peng, 2008, World J Microbiol. Biotechnol., 24(9), 1915-1922).

However, the fact of maintaining growth at its maximum rate (μ, in h⁻¹)is not always correlated with high production of the desired product.

In fact, it quickly became apparent to the specialists in the fieldthat, in order for microalgae to produce large lipid reserves, it isnecessary, for example, to subject the microalgae to a nutritionalstress that limits their growth.

Now, growth and production are decoupled in fermentation methods.

For example, to promote the accumulation of polyunsaturated fatty acids(here docosahexaenoic acid, or DHEA), patent application WO 01/54510advises to dissociate cell growth and polyunsaturated fatty acidproduction.

In the microalga Schizochytrium sp. strain ATCC 20888, a first growthphase without oxygen limiting is thus carried out so as to promote theproduction of a high cell density (greater than 100 g/l); then, in asecond step, the oxygen supply is gradually reduced so as to stress themicroalga, to slow its growth and to initiate the production of thefatty acids of interest.

In the microalga Crypthecodinium cohnii, the highest docosahexaenoicacid (DHEA, polyunsaturated fatty acid) content is obtained with a lowglucose concentration (about 5 g/l), and thus with low a growth rate(Jiang and Chen, 2000, Process Biochem., 35(10), 1205-1209).

These results illustrate clearly that the kinetics of product formationcan be positively or negatively associated with microalgae growth, andeven a combination of the two.

Consequently, in the case where product formation is not correlated withhigh cell growth, it is wise to control the cell growth rate.

In general, the skilled person chooses to control microalgae growth bycontrolling the fermentation conditions (Tp, pH, etc.) or by controllingthe supply of nutritional components to the fermentation medium(semi-continuous so-called “fed-batch” conditions).

If the skilled person chooses to control microalgae growth underheterotrophic conditions by means of the supply of carbon sources, he orshe generally chooses to adapt the carbon source (pure glucose, acetate,ethanol, etc.) to the microalga (C. cohnii, Euglena gracilis, etc.)according to the metabolite produced (a DHEA-type polyunsaturated fattyacid, for example).

The temperature may be a key parameter as well:

-   -   it has been reported, for example, that the synthesis of        polyunsaturated fatty acids by certain microalgae species, such        as EPA by Chlorella minutissima, is promoted at a temperature        lower than that required for the optimal growth of said        microalga;    -   conversely, the yield of lutein by Chlorella protothecoides        grown heterotrophically is higher when the production        temperature is increased from 24° C. to 35° C.

Chlorella protothecoides is justifiably recognized as one of the bestoil-producing microalgae.

Under heterotrophic conditions, it quickly transforms carbohydrates intotriglycerides (greater than 50% of its dry matter).

To optimize this triglyceride production, the skilled person is led tooptimize the carbon flux toward the production of oil by acting on thenutritional environment of the fermentation medium.

It is thus known that oil accumulates when a sufficient amount of carbonis supplied, but under nitrogen-deficient conditions.

The C/N ratio is thus decisive here, and it is acknowledged that thebest results are obtained by acting directly on the nitrogen content, asthe glucose content is not limiting.

Not surprisingly, this nitrogen deficiency affects cell growth,resulting in a growth rate that is 30% lower than the normal microalgalgrowth rate (Xiong et al., Plant Physiology, 2010, 154, pp. 1001-1011).

To explain this result, Xiong et al., in the above-mentioned article,show that, in fact, if the Chlorella biomass is divided into its fiveprincipal components, i.e., carbohydrates, lipids, proteins, DNA and RNA(representing 85% of its dry matter), if the C/N ratio has no impact onthe DNA, RNA and carbohydrate content, it becomes preeminent for theprotein and lipid content.

Thus it is that Chlorella cells grown with a low C/N ratio contain 25.8%proteins and 25.23% lipids, while a high C/N ratio enables the synthesisof 53.8% lipids and 10.5% proteins.

To optimize oil production, it is thus essential for the skilled personto control the carbon flux by diverting it toward oil production, to thedetriment of protein production; the carbon flux is redistributed andaccumulates in lipid storage substances when the microalgae are placedin nitrogen-deficient medium.

Armed with this teaching, to produce protein-rich biomasses the skilledperson is thus led to carry out the opposite of this metabolic control,i.e., to apply fermentation conditions that promote a low C/N ratioinstead, and thus:

-   -   to supply to the fermentation medium a large amount of the        nitrogen source, while maintaining constant the load of the        carbon source that will be converted into proteins; and    -   to stimulate the growth of the microalga.

It is a matter of modifying the carbon flux toward the production ofproteins (and thus of biomasses), to the detriment of the production ofstorage lipids.

In the context of the invention, the Applicant company chose to explorea novel pathway by disclosing an alternative solution to thattraditionally envisaged by the skilled person.

Thus, the invention relates to a method of protein enrichment of amicroalga grown heterotrophically, said microalga being of the genusChlorella, more particularly Chlorella sorokiniana or Chlorellaprotothecoides, said heterotrophic culturing method comprising a stepaiming at limiting the growth of said microalga by means of a deficiencyin the fermentation medium of a non-nitrogenous nutritional source.

This step is a heterotrophic culturing step where a non-nitrogenousnutritive factor is supplied in the medium in an amount that isinsufficient to allow the microalga to grow. It should be noted that“amount that is insufficient” does not mean that this nutritive factoris not supplied. The result of this nutrient-deficient phase is to slow(to limit) cell metabolism, without inhibiting it completely.

In the meaning of the invention, “enrichment” refers to an increase inthe protein content of the biomass of at least 15%, preferably at least20% by weight, so that the protein content of the biomass reaches morethan 50% by weight.

The invention covers more specifically a method of heterotrophicculturing of said microalgae comprising a step aiming at limiting thegrowth of said microalga by means of a deficiency in the fermentationmedium of a non-nitrogenous nutritional source.

The present invention thus relates to a method of protein enrichment ofa microalga grown heterotrophically, said microalga being of the genusChlorella, more particularly Chlorella sorokiniana or Chlorellaprotothecoides, the method comprising heterotrophic culturing thatincludes a step aiming at limiting the growth of said microalga by meansof a deficiency in the fermentation medium of a non-nitrogenousnutritional source, thus enabling the biomass protein content to reachmore than 50% by weight.

By “deficiency in the fermentation medium of a non-nitrogenousnutritional source” is meant a culture wherein at least one of thenon-nitrogenous nutritive factors is supplied to the microalga in anamount that is insufficient for it to grow.

In particular, the nutritional source is deficient so as to obtain agrowth rate that is 10% to 60% lower than the growth rate with nolimiting of said nutritional source. In particular, it is disclosed todecrease the growth rate by 10% to 60% compared with the growth ratewith no limiting of glucose, in particular by 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50% or 55% compared with the growth rate with no limitingof glucose. Preferably, the growth rate is decreased by 15% to 55%.

The duration of the culturing phase comprising a deficiency of anutritive factor, in particular of glucose, is at least 1 h, preferablyat least 10 h, more preferably at least 20 h, in particular between 30 hand 60 h.

The result is an absence of residual non-nitrogenous nutritive factor inthe culture medium, the microalga consuming this nutritive factor asfast as it is supplied. However, the absence of residual non-nitrogenousnutritive factor in the culture medium is distinguished from a situationin which the microalga is completely deprived of the nutritive factor.

In the context of the invention, the essential criterion is thus thelimiting of cell growth induced by stress, namely cellular stress causedby the deficiency in the fermentation medium of a non-nitrogenousnutritive substance.

This strategy is thus quite counter to the technical prejudice accordingto which, in order to increase the protein content of the biomass, it isinevitably necessary to increase the biomass and thus increase cellgrowth.

By “a non-nitrogenous nutritional source” is meant a nutritive substanceselected, for example, from the group consisting of glucose andphosphates.

As will be exemplified below, it may be advantageously chosen to limitthe growth of:

-   -   Chlorella sorokiniana by means of a glucose deficiency in the        fermentation medium,    -   Chlorella protothecoides by means of a phosphate deficiency in        the fermentation medium.

In particular, for these strains, the nutritive substance is supplied soas to obtain a growth rate of between 0.06 h⁻¹ and 0.09 h⁻¹.

In a very specific embodiment, the Chlorella sorokiniana strain is thestrain UTEX 1663 (The Culture Collection of Algae at the University ofTexas at Austin, USA). In a very specific embodiment, the Chlorellaprotothecoides strain is the strain CCAP211/8D (The Culture Collectionof Algae and Protozoa, Scotland, UK).

Optionally, the growth of said microalga can be limited by adding in theculture medium substances that inhibit cell growth, such as sulfates.

Furthermore, without being bound to any one theory, the Applicantcompany has found that the glucose flux in microalgae of the genusChlorella sorokiniana is normally used according to a quite preciseprioritization:

-   -   1. basal metabolism,    -   2. growth, i.e., formation of a protein-rich biomass,    -   3. storage substances (lipids and carbohydrates such as starch).

This principle explains the natural variations in protein content as themicroalga grows, despite the constant supply of nitrogen.

The Applicant company has thus found that in order to enrich themicroalgal biomass in proteins it is necessary to limit the growth ofthe microalga and to control its consumption of nutritive sources otherthan nitrogen, for example glucose, so as to:

-   -   dedicate the entire consumption of glucose to protein production        pathways,    -   avoid the accumulation of storage substances such as lipids.

Indeed, avoiding a nitrogen deficiency makes it possible to prevent thediversion of metabolic fluxes toward the production of lipids.

Optionally, it may be advantageous to go so far as to completely blockany synthesis of storage material, by the use of specific inhibitors.

Indeed, a certain number of inhibitors of the synthesis pathway forlipids, and even for starch (the storage carbohydrate par excellence ofgreen microalgae), are known:

-   -   for lipids, cerulenin is described as an inhibitor of fatty acid        synthesis, and lipstatin, a natural substance produced by        Streptomyces toxytricini, as an inhibitor of lipases, etc.;    -   for starch, iminosugars (obtained by simple substitution of the        endocyclic oxygen atom of sugars with a nitrogen atom) are        historically known as powerful inhibitors of glycosidases,        glycosyltransferases, glycogen phosphorylases and UDP-Galp        mutase.

Thus, the method comprises the fermentation of a microalgal biomassunder heterotrophic conditions with a first step of growing the biomassand a second step of depriving the fermentation medium of anon-nitrogenous nutritional source.

This second step makes it possible to enrich the biomass in protein. Inparticular, it makes it possible for the protein content of the biomassto reach more than 50% by weight (by weight of dry matter).

In a first preferred embodiment in accordance with the invention, themethod for heterotrophically culturing said microalgae, in particularChlorella sorokiniana, comprises:

-   -   a first step in which the microalgae are grown, where the        glucose supply rate is adjusted to the consumption capacity of        said microalgae so as to obtain a large biomass quickly,    -   a second step in which the microalgae produce proteins, where        the glucose supply rate is set to a value distinctly below the        glucose consumption capacity of said microalgae so as to prevent        the additional accumulation of storage substances or to promote        their consumption.

As will be exemplified below, the first step in which the microalgae aregrown is carried out in a discontinuous or “batch” mode in which theentirety of the initial glucose supply is consumed by the microalga,resulting in the production of a base biomass.

The second step in which proteins are produced is carried out underconditions in which:

-   -   either: complete medium is supplied semi-continuously, using a        so-called “fed-batch” system, after the initial glucose supply        is consumed; the other operational parameters of the        fermentation remain unchanged.

Glucose is supplied continuously and the supply rate is then lower thanthe strain's maximum rate of consumption so that the residual glucosecontent in the medium is kept at zero.

The strain's growth is thus limited by the availability of glucose(glucose-limiting condition).

-   -   or: a continuous chemostat operation is used, in which the        strain's growth rate (μ) is kept at its minimum value, the        strain's growth being limited by the glucose supply.

This method of operation makes it possible to obtain a biomass with ahigh protein content thanks to the limiting of glucose and to the lowgrowth rate imposed, all while ensuring very good productivity.

In a second preferred embodiment in accordance with the invention, themethod for heterotrophically culturing said microalgae, in particularChlorella protothecoides, comprises a step in which the microalgae aregrown, where a limiting of the phosphate supply limits the growth rateand results in an increase in the protein content. Thus, theheterotrophic culturing of microalgae of the species Chlorellaprotothecoides comprises a step of heterotrophic culturing with adeficiency of phosphates, the growth rate thus being reduced andresulting in an increase in the protein content.

The invention will be better understood with the aid of the followingexamples, which are meant to be illustrative and non-limiting.

EXAMPLES Example 1 Production of Chlorella sorokiniana UsingSequential-Batch Fermentation without Limiting the Supply of NutritiveMedium

The strain used is a Chlorella sorokiniana (strain UTEX 1663—The CultureCollection of Algae at the University of Texas at Austin, USA).

Preculture:

-   -   600 ml of medium in a 2 l Erlenmeyer flask;    -   Composition of the medium (Table 1 below)

TABLE 1 Macroelements Glucose 20 (g/l) K₂HPO₄•3H₂O 0.7 MgSO₄•7H₂O 0.34Citric acid 1.0 Urea 1.08 Na₂SO₄ 0.2 Na₂CO₃ 0.1 Clerol FBA 3107(defoamer) 0.5 Microelements Na₂EDTA 10 (mg/l) CaCl₂•2H₂O 80 FeSO₄•7H₂O40 MnSO₄•4H₂O 0.41 CoSO₄•7H₂O 0.24 CuSO₄•5H₂O 0.24 ZnSO₄•7H₂O 0.5 H₃BO₃0.11 (NH₄)₆Mo₇O₂₇•4H₂O 0.04

The pH is adjusted to 7 before sterilization by adding 8 N NaOH.

Incubation proceeds under the following conditions:

-   -   duration: 72 h;    -   temperature: 28° C.;    -   shaking speed: 110 rpm (Infors Multitron incubator).

The preculture is then transferred to a 30 l Sartorius fermentor.

Biomass Production Culture:

The medium is identical to that of the preculture, but the urea isreplaced with NH₄Cl.

TABLE 2 Macroelements Glucose 20 (g/l) K₂HPO₄•3H₂O 0.7 MgSO₄•7H₂O 0.34Citric acid 1.0 NH₄Cl 1.88 Na₂SO₄ 0.2 Clerol FBA 3107 (defoamer) 0.5Microelements Na₂EDTA 10 (mg/l) CaCl₂ 80 FeSO₄•7H₂O 40 MnSO₄•4H₂O 0.41CoSO₄•7H₂O 0.24 CuSO₄•5H₂O 0.24 ZnSO₄•7H₂O 0.5 H₃BO₃ 0.11(NH₄)₆Mo₇O₂₇•4H₂O 0.04

The initial volume (V_(i)) of the fermentor is adjusted to 13.5 l afterinoculation.

It is increased to a final volume of 16-20 l.

The operational parameters of the fermentation are as follows:

TABLE 3 Temperature 28° C. pH 5.0-5.2 using 28% NH₃ (w/w) pO₂ >20%(maintained by shaking) Shaking speed Minimum 300 rpm Air flow rate 15l/min

When the initially supplied glucose is consumed, a medium identical tothe initial medium, without the defoamer, is supplied in the form of aconcentrated solution containing 500 g/l of glucose and—in the sameproportions relative to glucose as in the initial medium—the otherelements, so as to obtain in the fermentor a glucose content of 20 g/l.

Two other identical additions are supplied in the same manner each timethe residual glucose concentration becomes zero.

Clerol FBA 3107 defoamer is added as needed to avoid excessive foaming.

Results:

After 46 h of culturing, 38 g/l of biomass is obtained with a proteincontent (evaluated by N 6.25) of 36.2%.

Example 2 Production of C. sorokiniana Using Fed-Batch Fermentation witha Limiting Supply of Glucose

In this example, a supply of complete medium (fed-batch mode) is startedafter the initially supplied glucose is consumed. The other operationalparameters of the fermentation remain unchanged.

Glucose is supplied continuously using a 500 g/l concentrated solution.The supply rate is lower than the strain's maximum consumption rate sothat the residual glucose content in the medium is kept at zero, i.e.,the strain's growth is limited by the availability of glucose(glucose-limiting condition).

This rate increases exponentially over time. The formula used tocalculate the addition flow rate is characterized by a factor μcorresponding to the growth rate that the strain can adopt if itconsumes all the glucose supplied:

S=So×exp (μ·t)

-   -   S=glucose supply flow rate (in g/h).    -   So=initial glucose supply flow rate, determined according to the        biomass present at the end of the batch. It is 12 g/h under our        conditions.    -   μ=flow rate acceleration factor. It should be below 0.11 h⁻¹,        which is the strain's growth rate in the absence of nutritional        limiting.    -   t=duration of the fed-batch operation (in h).

The salts are supplied continuously, if possible, separately or mixedwith glucose. But they may also be supplied sequentially in severalportions.

Table 4 below gives the salt requirements for 100 g of glucose.

TABLE 4 Macroelements Glucose 100 (g) K₂HPO₄•3H₂O 6.75 MgSO₄•7H₂O 1.7Citric acid 5.0 Na2SO4 1.0 Microelements Na₂EDTA 50 (mg) CaCl₂•2H₂O 400FeSO₄•7H₂O 200 MnSO₄•4H₂O 2.1 CoSO₄•7H₂O 1.2 CuSO₄•5H₂O 1.2 ZnSO₄•7H₂O2.5 H₃BO₃ 0.6 (NH₄)₆Mo₇O₂₇•4H₂O 0.2

The concentrations of the elements other than glucose were determined soas to be in excess relative to the strain's nutritional requirements.

Clerol FBA 3107 defoamer is added as needed to avoid excessive foaming.

Results: Effect of the Glucose Supply Rate During the Fed-BatchOperation

Tests were carried out at various glucose supply rates in fed-batchmode. They are characterized by the μ applied. The biomass proteincontent obtained is evaluated by measuring the total nitrogen expressedin N 6.25.

TABLE 5 μ fed Duration Biomass Productivity Test (h⁻¹) (h) (g/l) (g/l/h)% N 6.25 1 0.06 78 43.6 0.56 49.2 2 0.07 54 35.1 0.65 43.1 3 0.09 4864.9 1.35 39.3

These results show that glucose-limiting makes it possible to increasethe protein content.

Indeed, it is observed that, even with a high μ of 0.09, the proteincontent obtained is higher than that obtained without limiting, as inExample 1 (39.3% versus 36.2%).

Tighter limiting of the metabolism by means of glucose results in anadditional increase in protein content.

Under these test conditions, it is necessary to impose on the strain a μbelow 0.06 h⁻¹ in order to obtain a protein content above 50%.

It should be noted that this condition goes hand in hand with a decreasein productivity: 0.56 g/l/h versus 1.35 g/l/h in test 3.

Example 3 Production of C. sorokiniana Using Continuous ChemostatFermentation with a Limiting Supply of Glucose

In this example, a 2 l Sartorius Biostat B fermentor is used.

Fermentation is carried out as in Example 2, but with volumes 1/10 thesize: the volume of inoculum is 60 ml and the initial volume is 1.35 l.

The continuous supply of medium is started according to the sameprinciple as in Example 2, the salts being in this case mixed withglucose in the feed tank. The supply rate is accelerated according tothe same exponential formula as in Example 2 by applying a p of 0.06h⁻¹.

Chemostat

When a volume of 1.6 l is reached (a biomass concentration of about 50g/l), the continuous chemostat-type operation begins:

1. The fermentor is fed continuously at a flow rate of 96 ml/h with anutritive medium solution, containing 100 g/l of glucose, of thefollowing composition:

TABLE 6 Macroelements Glucose 100 (g/l) K₂HPO₄•3H₂O 6.75 MgSO₄•7H₂O 1.7Citric acid 5.0 Na₂SO4 1.0 Microelements Na₂EDTA 50 (mg/l) CaCl₂•2H₂O400 FeSO₄•7H₂O 200 MnSO₄•4H₂O 2.1 CoSO₄•7H₂O 1.2 CuSO₄•5H₂O 1.2ZnSO₄•7H₂O 2.5 H₃BO₃ 0.6 (NH₄)₆Mo₇O₂₇•4H₂O 0.2

The concentrations of the elements other than glucose were determined soas to be in excess relative to the strain's nutritional requirements.

2. Medium is removed continuously from the fermentor by means of asiphon tube connected to a pump, so as to keep the culture volume at 1.6l.

Thus, a 0.06/1 fraction (6%) of the medium is replaced per hour. Thisreplacement rate is called the dilution rate (D).

In accordance with the principle of the chemostat culturing method, thestrain's growth rate (μ) is established at the same value because thestrain's growth is limited by the glucose supply:

D=μ=0.06 h⁻¹

Results

After 97 h of operation in chemostat mode, the biomass concentrationstabilizes at 48 g/l±2 g/l and the protein content at 53±2%.

This mode of operation makes it possible to obtain a biomass with a highprotein content thanks to the limiting of glucose and to the low growthrate imposed, all while ensuring very good productivity, of about 2.9g/l/h, thanks to the high concentration of biomass.

Example 4 Production of Chlorella protothecoides Using BatchFermentation with or without Limiting of the Phosphate Supply

The strain used is Chlorella protothecoides (strain CCAP21 1/8D—TheCulture Collection of Algae and Protozoa, Scotland, UK).

Preculture:

-   -   150 ml of medium in a 500 ml Erlenmeyer flask;    -   Composition of the medium: 40 g/l of glucose+10 g/l of yeast        extract.

Incubation proceeds under the following conditions: duration: 72 h;temperature: 28° C.; shaking speed: 110 rpm (Infors Multitronincubator).

The preculture is then transferred to a 2 l Sartorius Biostat Bfermentor.

Biomass Production Culture:

The composition of the culture medium is as follows (in g/l):

TABLE 7 Glucose 80 Citric acid 4 NH₄CL 2 KH₂PO₄ 2 (test 1) or 3 (test 2)Na₂HPO₄ 2 (test 1) or 3 (test 2) MgSO₄, 7H₂O 1.5 NaCl 0.5 Yeast extract5

The phosphate supply is calculated so as to be limiting in test 1 and inexcess in test 2. Clerol FBA 3107 defoamer is added as needed to avoidexcessive foaming. The initial volume (V_(i)) of the fermentor isadjusted to 1 l after inoculation.

The operational parameters of the fermentation are as follows:

TABLE 8 Temperature 28° C. pH 6.5 using 28% NH₃ (w/w) pO₂ >20%(maintained by shaking) Shaking speed Minimum 200 rpm Air flow rate 1l/min

Results:

TABLE 9 Duration Biomass Cumulative Residual % N Test (h) (g/l) μ (h⁻¹)PO₄ (mg/l) 6.25 1 45 36.5 0.07 0 56.1 2 36 38.1 0.09 800 48.1

These results show that a limiting of the phosphate supply, confirmed bythe absence of residual phosphate at the conclusion of the fermentation,limits the growth rate (measured by the cumulative μ) and, like thelimiting of glucose in the preceding examples, results in an increase inthe protein content reaching values distinctly greater than 50%.

1-8. (canceled)
 9. A method of protein enrichment of a microalga grownheterotrophically, said microalga being of the genus Chlorella,comprising a step of limiting the growth of said microalga by means of adeficiency in the fermentation medium of a non-nitrogenous nutritionalsource, thus making it possible for the biomass protein content to reachmore than 50% by weight.
 10. The method of claim 9, wherein thedeficient non-nitrogenous nutritional source is selected from the groupconsisting of glucose and phosphates.
 11. The method of claim 10,wherein the non-nitrogenous nutritional source is deficient so as toobtain a growth rate that is 10% to 60% lower than the growth ratewithout the limiting of said non-nitrogenous nutritional source.
 12. Themethod of claim 11, wherein the microalgae of the genus Chlorella areselected from the group consisting of Chlorella sorokiniana andChlorella protothecoides.
 13. The method of claim 12, wherein saidmicroalgae are supplied with a nutritive substance so as to obtain agrowth rate of between 0.06 h⁻¹ and 0.09 h⁻¹.
 14. The method of claim 9,wherein the duration of the deficient culturing phase is at least 1 h.15. The method of claim 9, wherein the duration of the deficientculturing phase is at least 10 h.
 16. The method of claim 9, wherein theduration of the deficient culturing phase is at least 20 h.
 17. Themethod of claim 9, wherein the duration of the deficient culturing phaseis between 30 h and 60 h.
 18. The method of claim 12, wherein theheterotrophic culturing of microalgae of the species Chlorellasorokiniana comprises: a) a first step in which the microalgae are grownand the glucose supply rate is adjusted to the consumption capacity ofsaid microalgae so as to obtain a large biomass quickly, and b) a secondstep in which the microalgae produce proteins, where the glucose supplyrate is set to a value distinctly below the glucose consumption capacityof said microalgae so as to prevent the additional accumulation ofstorage substances or to promote their consumption.
 19. The method ofclaim 12, wherein the heterotrophic culturing of microalgae of thespecies Chlorella protothecoides comprises a step of culturing with aphosphate deficiency that limits the growth rate and results in anincrease in the protein content.